The sarcomere is the basic contractile unit in striated muscle. While mutations in sarcomeric proteins have been linked to various human cardiovascular diseases, it is unclear why mutations in different sarcomeric genes or even different mutations in the same gene result in distinct diseases. The goal of this proposal is to use titin as a paradigm to investigate molecular mechanisms of sarcomere assembly and sarcomeric diseases. We have identified six titin mutants in zebrafish from mutagenesis screens and demonstrated that phenotypes from one mutant allele resemble human dilated cardiomyopathy. We recently identified the full-length genomic sequence for zebrafish titin (tinl) and cloned the other titin homologue (tin2) in zebrafish. Our preliminary data support the central hypothesis of this proposal, which predicts that different domains of Titin participate in distinct steps of myofibrillogenesis disruption of which results in different sarcomeric diseases. In our Specific Aim 1, we will investigate the functional divergence of tinl and tin2. By leveraging morpholino technology, we will test the hypothesis that different isoforms of titin have different functions in myofibrillogenesis. In our Specific Aim 2, we will test the hypothesis that different domains of titin have different functions in myofibrillogenesis. We will generate a series of truncations of both tinl and tin2 in vivo to reveal the functions of the C-terminal domains, including the kinase domain. We will also investigate the function of a Titin N-terminal domain by knocking out a titin interacting protein, Tcap. In our Specific Aim3, we will determine the pathological consequences of different titin mutations in zebrafish. We will first identify the location of mutations in all six titin mutants and then characterize the pathological phenotypes in both embryonic and adult fish. By generating and characterizing a series of titin mutations, these experiments will deepen our understanding of why mutations in different titin exons result in distinct structural changes of the sarcomere and different phenotypes of sarcomeric diseases, including dilated cardiomyopathy, hypertrophic cardiomyopathy, and muscular dystrophy.